The widespread use of the prostate-specific antigen (PSA) test had a great impact on the rise of incidence of prostate cancer. The magnitude of opportunistic screening by PSA varies across countries, being highest in the US, and considerably lower in Europe. To estimate the opportunistic use of PSA over the period of one year (2000) in the District of Florence, we analysed the Regional Database of diagnostic exams. According to the Regional Database, subjects having at least one PSA test during the year 2000 ranged from 5.8 to 6.7%, 8.8–10.8%, 11.7–15.0%, 16.5–22.3%, 18.0–24.3%, 17.0–23.8% and 14.1–18.3% in the 50–54, 55–59, 60–64, 65–69, 70–74, 75–79 and >79 years age groups, respectively. Minimum and maximum values are reported according to exclusion or inclusion of PSA determinations with incomplete subject identification data. Such a high use of PSA should deserve special attention from both the medical and health care provider community. The aim of the present study was to evaluate the frequency of opportunistic screening by PSA in the District of Florence in the general population.